Abnormal Paneth Cells Research Articles

Interplay of gut microbiota and host epithelial mitochondrial dysfunction is necessary for the development of spontaneous intestinal inflammation in mice

BackgroundIntestinal epithelial cell (IEC) mitochondrial dysfunction involvement in inflammatory bowel diseases (IBD), including Crohn’s disease affecting the small intestine, is emerging in recent studies. As the interface between the self and the gut microbiota, IECs serve as hubs of bidirectional cross-talk between host and luminal microbiota. However, the role of mitochondrial-microbiota interaction in the ileum is largely unexplored. Prohibitin 1 (PHB1), a chaperone protein of the inner mitochondrial membrane required for optimal electron transport chain function, is decreased during IBD. We previously demonstrated that mice deficient in PHB1 specifically in IECs (Phb1i∆IEC) exhibited mitochondrial impairment, Paneth cell defects, gut microbiota dysbiosis, and spontaneous inflammation in the ileum (ileitis). Mice deficient in PHB1 in Paneth cells (epithelial secretory cells of the small intestine; Phb1∆PC) also exhibited mitochondrial impairment, Paneth cell defects, and spontaneous ileitis. Here, we determined whether this phenotype is driven by Phb1 deficiency-associated ileal microbiota alterations or direct effects of loss of PHB1 in host IECs.ResultsDepletion of gut microbiota by broad-spectrum antibiotic treatment in Phb1∆PC or Phb1i∆IEC mice revealed a necessary role of microbiota to cause ileitis. Using germ-free mice colonized with ileal microbiota from Phb1-deficient mice, we show that this microbiota could not independently induce ileitis without host mitochondrial dysfunction. The luminal microbiota phenotype of Phb1i∆IEC mice included a loss of the short-chain fatty acid butyrate. Supplementation of butyrate in Phb1-deficient mice ameliorated Paneth cell abnormalities and ileitis. Phb1-deficient ileal enteroid models suggest deleterious epithelial-intrinsic responses to ileal microbiota that were protected by butyrate.ConclusionsThese results suggest a mutual and essential reinforcing interplay of gut microbiota and host IEC, including Paneth cell, mitochondrial health in influencing ileitis. Restoration of butyrate is a potential therapeutic option in Crohn’s disease patients harboring epithelial cell mitochondrial dysfunction.BykqUrdWkjvtZT9W9KFZmHVideo

Reduced γδ intraepithelial lymphocyte number and motility precede the onset of Crohn’s disease-like ileitis.

Abstract Crohn’s disease (CD) is a relapsing, remitting disease resulting in chronic intestinal inflammation. Recent studies of resected ileal tissue from CD patients reported a loss of intraepithelial lymphocytes expressing the γδ T cell receptor (γδ IELs). γδ IELs continuously survey the epithelial barrier and protect against experimental colitis; however, the role of γδ IELs in the initiation of ileitis is unknown. To this end, we profiled the IEL compartment in TNF ΔARE/+mice, which develop chronic CD-like ileitis at 8 wks old, prior to and during ileitis onset (4–10 wks). We observed a 2.5-fold reduction in the frequency of ileal γδ IELs in 5-wk-old TNF ΔARE/+mice compared to TNF +/+(WT) littermates (p<0.001). Using intravital microscopy, we observed reduced γδ IEL surveillance behavior (10.6 vs 6.4 mm/min track speed mean, p<0.0001) in TNF ΔARE/+mice compared to WT at 6 wks. Analysis of cleared ileal tissue from 6-wk-old WT and TNF ΔARE/+mice revealed a reduction in the number of γδ IELs in villus tips in TNF ΔARE/+mice relative to WT, thereby increasing the proportion of γδ IELs found in the crypt region. Based on several reported links between γδ IELs and Paneth cells (PC), we quantified PCs by lysozyme immunostaining in ileal tissue. We found an increased proportion of morphologically abnormal PCs in 6-wk-old TNF ΔARE/+mice, reflecting PC dysfunction consistent with CD pathology. These findings are supported by a reduction in ileal Lyz1 expression in TNF ΔARE/+mice compared to WT prior to ileitis onset (p=0.02). Together, these studies demonstrate that γδ IELs exhibit impaired surveillance behavior and altered distribution along the crypt/villus axis prior to ileitis onset, which coincides with PC dysfunction in CD-like ileitis. COCR23PRF017 NJCCR Predoctoral Fellowship Golovchenko (PI) June 1, 2022 – May 31, 2024 γδ IEL surveillance behavior in the onset of chronic ileitis

P-112 Oleic acid promotes the malignant transformation of Kras-mutant colonic organoids via the expansion of tumorigenic stem cells and abnormal Paneth cells through upregulation of NFATc family

Although the five-year survival rate of colorectal cancer (CRC) is significantly high with localized stage (90%), only 38% of the patients are diagnosed at this stage. Therefore, finding high risk factors of CRC could prevent the disease at the early stage. Obesity and high-fat diet consumption are risk factors of CRC. In detail, oleic acids (OA) were found to accumulate in the adipose tissues of obese patients as well as being the most common long chain fatty acid in dietary lipid. The role of OA in colorectal cancer development is still controversary. On the other hand, organoids, which can be derived from adult tissue stem cells, are the miniatures of organs. Recent works suggest organoids are useful tools to dissect mechanisms involved in cancer development. The current work aims to utilize crypt stem cell-derived organoids to dissect how obesity can contribute to CRC development. Organoids were derived from Lgr5 + -EGFP mice (normal colon control), Lgr5 + -EGFP-creER; LSL-KrasG12D mice (colonic epithelium harbored KrasG12D mutation) and AOM/DSS-induced CRC mice (cancerous colon control). For in-vitro, the impact of OA on the malignant transformation of Kras-mutant colonic organoids was assessed by MTT proliferation assay, 2D transwell migration and invasion assay, irinotecan-resistant test and colony formation assay. The tumor formation in-vivo was induced by subcutaneous engrafting of OA-treated organoids on NOD/SCID mice. RNAseq was done to screen some differential expressed genes when treating OA. The existence of stem cell and abnormal Paneth cell population was demonstrated by immunostaining, flow cytometry and ELISA. OA enhanced the malignant of Kras-mutant colonic organoids through increasing proliferation, migration, invasion, drug resistance and colony formation. Besides, mice which were engrafted with OA-trained Lgr5-Kras organoids possessed larger tumors. The colonic stem cell population which were represented by positive Lgr5-GFP + cells were upregulated in OA-treated group. Interestingly, there is an existence of abnormal Lyz + /CD24 + Paneth cells in OA-treated Kras-mutant organoids. This suggested that OA may promote abnormal differentiation of colonic progenitor cells. Indeed, treatment of OA upregulated ATOH1 and GFI1, which contributed to select between goblet/Paneth and enteroendocrine cell fates. Besides, NFAT family, among which NFATc3 expressed the most, significantly increased in OA-treated organoids. The upregulation of NFATc was induced by inhibition of proteasomal degradation resulting from low expression of the complex RNF123, USP18 and TRIM30A. In addition, inhibition of the nuclear translocalization of NFATc family by VIVIT demonstrated the decline of stemness markers as well as Paneth cell markers. Finally, we showed the role of NFATc, especially NFATc3, in promoting the malignancy of OA-treated Kras-mutant organoids. Our findings suggested the impact of oleic acid on promoting the malignant transformation of Kras-mutant colonic organoids through NFATc-relating pathway. The expansion of tumorigenic stem cells via induction of abnormal Paneth cell, may contribute to the potential of cancer-toward metaplasia in Kras-mutant organoids when exposing to high level of oleic acid. Oleic acid is an unfavorable factor of colorectal cancer. The frequent usage of OA, which may benefit to cardiovascular diseases, may lead to the increased risk of CRC in latent sensitive Kras-mutant individuals.

AN AUTOPHAGY PROTEIN ATG16L1 PREVENTS NECROPTOSIS BY REGULATING INTERFERON SIGNALING IN THE INTESTINAL EPITHELIUM

ATG16L1, an essential component of autophagy, is one of the susceptibility genes of Crohn’s disease. We previously showed mice with decreased expression of Atg16L1 and Crohn’s disease patients homozygous for ATG16L1T300A risk alleles develop Paneth cell abnormalities in the intestinal epithelium. Moreover, the abnormalities were dependent on murine norovirus (MNV) infection in mice. Most recently, we reported mice with deletion of ATG16L1 in the intestinal epithelial cells (Atg16L1ΔIEC) are more susceptible to disease following intestinal injury than wild type mice when they are infected with MNV. Also, we revealed intestinal organoids derived from Atg16L1ΔIEC mouse are susceptible to TNFa-induced necroptosis. However, how ATG16L1 protects IECs against TNFa, and whether the susceptibility is preserved in human IECs harboring ATG16L1T300A risk alleles remains unclear.

AN AUTOPHAGY PROTEIN ATG16L1 PREVENTS NECROPTOSIS BY REGULATING INTERFERON SIGNALING IN THE INTESTINAL EPITHELIUM

Abstract Introduction ATG16L1, an essential component of autophagy, is one of the susceptibility genes of Crohn’s disease. We previously showed mice with decreased expression of Atg16L1 and Crohn’s disease patients homozygous for ATG16L1T300A risk alleles develop Paneth cell abnormalities in the intestinal epithelium. Moreover, the abnormalities were dependent on murine norovirus (MNV) infection in mice. Most recently, we reported mice with deletion of ATG16L1 in the intestinal epithelial cells (Atg16L1ΔIEC) are more susceptible to disease following intestinal injury than wild type mice when they are infected with MNV. Also, we revealed intestinal organoids derived from Atg16L1ΔIEC mouse are susceptible to TNFa-induced necroptosis. However, how ATG16L1 protects IECs against TNFa, and whether the susceptibility is preserved in human IECs harboring ATG16L1T300A risk alleles remains unclear. Aims In this study, we first examined the mechanism by which ATG16L1-deficiency renders IECs susceptible to necroptosis. Next, we investigated whether a specific ISG might be involved in the increased susceptibility. Finally, we tested whether human intestinal organoids harboring ATG16L1T300A risk alleles are susceptible to TNFa-induced necroptosis. Methods & Results We performed RNA-Seq analysis using small intestinal organoids derived from Atg16L1ΔIEC and control mouse, and found 49 genes were upregulated in naïve Atg16L1ΔIEC organoids (Figure 1). Interestingly, most of them were the genes known as interferon-stimulated genes (ISGs) representing a type I IFN (IFN-I) signature. Importantly, we found Atg16L1ΔIEC organoids pretreated with JAK-STAT inhibitor Ruxolitinib displayed better viability and decreased level of necroptosis-related proteins when they were stimulated with TNFa. Moreover, we found protein kinase R (PKR) was increased in naïve Atg16L1ΔIEC organoids, and inhibition of PKR significantly protected Atg16L1ΔIEC organoids from TNFa. Finally, we generated human intestinal organoids using biopsy specimens mostly collected from Crohn’s disease patients. We compared the viability between individuals with 2 copies vs 0 or 1 copy of ATG16L1T300A risk allele, and found organoids derived from ATG16L1T300A homozygous individuals exhibited heightened susceptibility to TNFa (Figure 2 A and B). Moreover, we tested the effect of Ruxolitinib as well as multiple necroptosis inhibitors on human organoids, and found all the inhibitors successfully protected ATG16L1T300A homozygous organoids from TNFa-induced cell death. Conclusion Our data indicate ATG16L1 prevents necroptosis by regulating interferon signaling in both mouse and human IECs, and suggest inhibition of JAK-STAT as well as necroptosis pathway could be promising therapeutic options for Crohn’s disease patients with ATG16L1T300A risk alleles.

Paneth cell α-defensin misfolding correlates with dysbiosis and ileitis in Crohn’s disease model mice

Crohn's disease (CD) is an intractable inflammatory bowel disease, and dysbiosis, disruption of the intestinal microbiota, is associated with CD pathophysiology. ER stress, disruption of ER homeostasis in Paneth cells of the small intestine, and α-defensin misfolding have been reported in CD patients. Because α-defensins regulate the composition of the intestinal microbiota, their misfolding may cause dysbiosis. However, whether ER stress, α-defensin misfolding, and dysbiosis contribute to the pathophysiology of CD remains unknown. Here, we show that abnormal Paneth cells with markers of ER stress appear in SAMP1/YitFc, a mouse model of CD, along with disease progression. Those mice secrete reduced-form α-defensins that lack disulfide bonds into the intestinal lumen, a condition not found in normal mice, and reduced-form α-defensins correlate with dysbiosis during disease progression. Moreover, administration of reduced-form α-defensins to wild-type mice induces the dysbiosis. These data provide novel insights into CD pathogenesis induced by dysbiosis resulting from Paneth cell α-defensin misfolding and they suggest further that Paneth cells may be potential therapeutic targets.

Mitochondrial dysfunction during loss of prohibitin 1 triggers Paneth cell defects and ileitis

ObjectiveAlthough perturbations in mitochondrial function and structure have been described in the intestinal epithelium of Crohn’s disease and ulcerative colitis patients, the role of epithelial mitochondrial stress in the pathophysiology...

Imbalance of autophagy and apoptosis in intestinal epithelium lacking the vitamin D receptor.

Apoptosis and autophagy are dynamic processes that determine the fate of cells. Vitamin D receptor (VDR) deficiency in the intestine leads to abnormal Paneth cells and impaired autophagy function. Here, we will elucidate the mechanisms of the intestinal epithelial VDR regulation of autophagy and apoptosis. We used in vivo VDRlox and VDR∆IEC mice and ex vivo organoids generated from small intestine and colon tissues. We found that VDR deficiency induced more apoptotic cells and significantly increased cell death in the small intestine and colon of VDR∆IEC mice. The proapoptotic protein B-cell lymphoma 2 (BCL-2) associated X protein (Bax) was enhanced, whereas autophagy related 16 like 1 (ATG16L1) and Beclin-1 were decreased in the intestines of VDRΔIEC mice. Apoptosis induced by Bax reduced autophagy by decreasing Beclin-1. Physical interactions between Beclin-1 and Bcl-2 were increased in the VDR-deficient epithelia from mice. The growth of VDR∆IEC organoids was significantly slower with fewer Paneth cells than that of VDR+/+ organoids. The expression levels of Beclin-1 and lysozyme were decreased in VDR∆IEC organoids. Bacterial endotoxin levels were high in the serum from VDR∆IEC mice and made mice susceptible to colitis. In the organoids and colitis IL-10-/- mice, vitamin D3 treatment increased VDR and ATG16L1 protein expression levels, which activated autophagic responses. In summary, intestinal epithelial VDR regulates autophagy and apoptosis through ATG16L1 and Beclin-1. Our studies provide fundamental insights into the tissue-specific function of VDR in modulating the balance between autophagy and apoptosis.-Lu, R., Zhang, Y.-G., Xia, Y., Sun, J. Imbalance of autophagy and apoptosis in intestinal epithelium lacking the vitamin D receptor.

14 AUTOPHAGY PROTEIN ATG16L1 PREVENTS NECROPTOSIS IN THE INTESTINAL EPITHELIUM.

Previous studies revealed both genetic and environmental factors contribute to the Crohn’s disease pathogenesis. ATG16L1, an essential component of autophagy, is one of the susceptibility genes of the disease. We previously showed that mice with decreased expression of ATG16L1 and Crohn’s disease patients homozygous for the ATG16L1 risk allele develop Paneth cell abnormalities in the intestinal epithelium, and the abnormalities were dependent on murine norovirus (MNV) infection. MNV is a single stranded RNA virus that persistently infects the gastrointestinal tract. We have recently showed that MNV can provide similar benefits to the host as bacteria. However, it is still unclear how this otherwise beneficial virus induces disease pathologies in the Atg16L1 mutant setting. In this study, we investigate the role of ATG16L1 in the intestinal epithelial cell (IEC) in virally triggered inflammatory disease. To investigate the role of intestinal ATG16L1, we treated IEC specific ATG16L1-deficient mice (Atg16L1ΔIEC) with dextran sodium sulfate (DSS) in the presence or absence of MNV. We observed their survival, clinical score, and histopathology. To further examine the mechanism by which ATG16L1 functions in IEC, we derived intestinal organoids from Atg16L1ΔIEC and control mice. Finally, we investigated the effect of MNV-infection on the intestinal intraepithelial lymphocytes (IELs), T cells within the epithelial layer of intestinal mucosa with cytolytic and immunoregulatory capacities. We found that Atg16L1ΔIEC mice were more susceptible to disease following intestinal injury when they were infected with MNV (Figure 1). Instead of alterations in viral replication, this susceptibility was associated with inflammatory cytokine production and exacerbated IEC death. Intestinal organoids derived from Atg16L1ΔIEC mice displayed smaller size, fewer budding, and impaired survival, which were further exaggerated by the addition of TNFa. Among the epithelial cell types, Paneth cells were the most vulnerable to cell death in ATG16L1-deficient organoids. Mechanistic analysis revealed that TNFa induced necroptosis (programmed necrosis) in the ATG16L1-deficient IEC, which was rescued by the inhibition of RIPK1 or RIPK3-deficiency. Moreover, we found that MNV-infection altered the IELs’ subpopulations and affected their motility in vivo, and in vitro co-culture system revealed that IELs harvested from MNV-infected host killed Atg16L1ΔIEC organoids compared with those from naïve host (Figure 2). Our findings support a disease model in which intestinal ATG16L1 prevents TNFa-induced necroptosis in the presence of commensal viruses in the gut, and also suggest that necroptosis inhibition as well as TNFa blockade could be a new therapeutic option for the Crohn’s disease patients with ATG16L1 risk allele.

14 AUTOPHAGY PROTEIN ATG16L1 PREVENTS NECROPTOSIS IN THE INTESTINAL EPITHELIUM

Previous studies revealed both genetic and environmental factors contribute to the Crohn’s disease pathogenesis. ATG16L1, an essential component of autophagy, is one of the susceptibility genes of the disease. We previously showed that mice with decreased expression of ATG16L1 and Crohn’s disease patients homozygous for the ATG16L1 risk allele develop Paneth cell abnormalities in the intestinal epithelium, and the abnormalities were dependent on murine norovirus (MNV) infection. MNV is a single stranded RNA virus that persistently infects the gastrointestinal tract. We have recently showed that MNV can provide similar benefits to the host as bacteria. However, it is still unclear how this otherwise beneficial virus induces disease pathologies in the Atg16L1 mutant setting. In this study, we investigate the role of ATG16L1 in the intestinal epithelial cell (IEC) in virally triggered inflammatory disease. To investigate the role of intestinal ATG16L1, we treated IEC specific ATG16L1-deficient mice (Atg16L1ΔIEC) with dextran sodium sulfate (DSS) in the presence or absence of MNV. We observed their survival, clinical score, and histopathology. To further examine the mechanism by which ATG16L1 functions in IEC, we derived intestinal organoids from Atg16L1ΔIEC and control mice. Finally, we investigated the effect of MNV-infection on the intestinal intraepithelial lymphocytes (IELs), T cells within the epithelial layer of intestinal mucosa with cytolytic and immunoregulatory capacities. We found that Atg16L1ΔIEC mice were more susceptible to disease following intestinal injury when they were infected with MNV (Figure 1). Instead of alterations in viral replication, this susceptibility was associated with inflammatory cytokine production and exacerbated IEC death. Intestinal organoids derived from Atg16L1ΔIEC mice displayed smaller size, fewer budding, and impaired survival, which were further exaggerated by the addition of TNFa. Among the epithelial cell types, Paneth cells were the most vulnerable to cell death in ATG16L1-deficient organoids. Mechanistic analysis revealed that TNFa induced necroptosis (programmed necrosis) in the ATG16L1-deficient IEC, which was rescued by the inhibition of RIPK1 or RIPK3-deficiency. Moreover, we found that MNV-infection altered the IELs’ subpopulations and affected their motility in vivo, and in vitro co-culture system revealed that IELs harvested from MNV-infected host killed Atg16L1ΔIEC organoids compared with those from naïve host (Figure 2). Our findings support a disease model in which intestinal ATG16L1 prevents TNFa-induced necroptosis in the presence of commensal viruses in the gut, and also suggest that necroptosis inhibition as well as TNFa blockade could be a new therapeutic option for the Crohn’s disease patients with ATG16L1 risk allele.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

The Crohn's disease polymorphism, ATG16L1 T300A, alters the gut microbiota and enhances the local Th1/Th17 response.

Inflammatory bowel disease (IBD) is driven by dysfunction between host genetics, the microbiota, and immune system. Knowledge gaps remain regarding how IBD genetic risk loci drive gut microbiota changes. The Crohn's disease risk allele ATG16L1 T300A results in abnormal Paneth cells due to decreased selective autophagy, increased cytokine release, and decreased intracellular bacterial clearance. To unravel the effects of ATG16L1 T300A on the microbiota and immune system, we employed a gnotobiotic model using human fecal transfers into ATG16L1 T300A knock-in mice. We observed increases in Bacteroides ovatus and Th1 and Th17 cells in ATG16L1 T300A mice. Association of altered Schaedler flora mice with B. ovatus specifically increased Th17 cells selectively in ATG16L1 T300A knock-in mice. Changes occur before disease onset, suggesting that ATG16L1 T300A contributes to dysbiosis and immune infiltration prior to disease symptoms. Our work provides insight for future studies on IBD subtypes, IBD patient treatment and diagnostics.

Integrative analysis of Paneth cell proteomic and transcriptomic data from intestinal organoids reveals functional processes dependent on autophagy.

ABSTRACTPaneth cells are key epithelial cells that provide an antimicrobial barrier and maintain integrity of the small-intestinal stem cell niche. Paneth cell abnormalities are unfortunately detrimental to gut health and are often associated with digestive pathologies such as Crohn's disease or infections. Similar alterations are observed in individuals with impaired autophagy, a process that recycles cellular components. The direct effect of autophagy impairment on Paneth cells has not been analysed. To investigate this, we generated a mouse model lacking Atg16l1 specifically in intestinal epithelial cells, making these cells impaired in autophagy. Using three-dimensional intestinal organoids enriched for Paneth cells, we compared the proteomic profiles of wild-type and autophagy-impaired organoids. We used an integrated computational approach combining protein-protein interaction networks, autophagy-targeted proteins and functional information to identify the mechanistic link between autophagy impairment and disrupted pathways. Of the 284 altered proteins, 198 (70%) were more abundant in autophagy-impaired organoids, suggesting reduced protein degradation. Interestingly, these differentially abundant proteins comprised 116 proteins (41%) that are predicted targets of the selective autophagy proteins p62, LC3 and ATG16L1. Our integrative analysis revealed autophagy-mediated mechanisms that degrade key proteins in Paneth cell functions, such as exocytosis, apoptosis and DNA damage repair. Transcriptomic profiling of additional organoids confirmed that 90% of the observed changes upon autophagy alteration have effects at the protein level, not on gene expression. We performed further validation experiments showing differential lysozyme secretion, confirming our computationally inferred downregulation of exocytosis. Our observations could explain how protein-level alterations affect Paneth cell homeostatic functions upon autophagy impairment.This article has an associated First Person interview with the joint first authors of the paper.

Environmental factors regulate Paneth cell phenotype and host susceptibility to intestinal inflammation in Irgm1-deficient mice.

ABSTRACTCrohn's disease (CD) represents a chronic inflammatory disorder of the intestinal tract. Several susceptibility genes have been linked to CD, though their precise role in the pathogenesis of this disorder remains unclear. Immunity-related GTPase M (IRGM) is an established risk allele in CD. We have shown previously that conventionally raised (CV) mice lacking the IRGM ortholog, Irgm1 exhibit abnormal Paneth cells (PCs) and increased susceptibility to intestinal injury. In the present study, we sought to utilize this model system to determine if environmental conditions impact these phenotypes, as is thought to be the case in human CD. To accomplish this, wild-type and Irgm1−/− mice were rederived into specific pathogen-free (SPF) and germ-free (GF) conditions. We next assessed how these differential housing environments influenced intestinal injury patterns, and epithelial cell morphology and function in wild-type and Irgm1−/− mice. Remarkably, in contrast to CV mice, SPF Irgm1−/− mice showed only a slight increase in susceptibility to dextran sodium sulfate-induced inflammation. SPF Irgm1−/− mice also displayed minimal abnormalities in PC number and morphology, and in antimicrobial peptide expression. Goblet cell numbers and epithelial proliferation were also unaffected by Irgm1 in SPF conditions. No microbial differences were observed between wild-type and Irgm1−/− mice, but gut bacterial communities differed profoundly between CV and SPF mice. Specifically, Helicobacter sequences were significantly increased in CV mice; however, inoculating SPF Irgm1−/− mice with Helicobacter hepaticus was not sufficient to transmit a pro-inflammatory phenotype. In summary, our findings suggest the impact of Irgm1-deficiency on susceptibility to intestinal inflammation and epithelial function is critically dependent on environmental influences. This work establishes the importance of Irgm1−/− mice as a model to elucidate host-environment interactions that regulate mucosal homeostasis and intestinal inflammatory responses. Defining such interactions will be essential for developing novel preventative and therapeutic strategies for human CD.

P109 SMOKING NEGATIVELY AFFECTS DISEASE COURSE REGARDLESS OF SMOKING AMOUNT AND MAY BE ASSOCIATED WITH PANETH CELL PHENOTYPE IN JAPANESE CROHN’S DISEASE PATIENTS

Smoking is a well-known risk factor for Crohn’s disease (CD) onset and aggressive disease course in the Western population. However, previous studies imply that smoking was not associated with an increased risk for CD onset in Asian populations. Therefore, we examined the effect of smoking on the disease course after surgery in Japanese CD patients. Furthermore, we investigated the association between smoking and paneth cell (PC) phenotype, which was recently recognized as a useful biomarker for the disease course of CD. A total of 72 Japanese CD patients who underwent ileal resection were recruited. Patents were classified into three groups by post-operative smoking status; current smoker：patients who continued smoking after surgery; ever smoker：patients who did not smoke after surgery but had smoking history; and non-smoker. Pre and post-operative smoking amount were investigated in current smokers. PC phenotype was determined by defensin 5 immunofluorescence on uninvolved ileal tissue. Correlation with time to recurrence was performed using the log-rank and cox hazard test. Current smoking were significantly associated with early recurrence compared with the other two groups (P=0.0063). There was no significant difference between ever smoker and non-smoker (P=0.643). Moreover, pre and post-operative smoking amount were not associated with recurrence time (P=0.438). Apart from current smoking, first use of biologics and PC phenotype were correlated with early recurrence (P=0.023, 0.0065). Interestingly, patients who were current smokers at the time of the operation tended to have higher proportion of abnormal PC phenotype (P=0.054). Smoking after surgery was shown to negatively affect the disease course regardless of the smoking amount in Japanese CD. Not reduced smoking but nonsmoking should be recommended for CD patients. Furthermore, smoking can affect PC phenotype, which may explain negative effect of smoking on CD.

LRRK2 but not ATG16L1 is associated with Paneth cell defect in Japanese Crohn's disease patients.

BACKGROUND. Morphological patterns of Paneth cells are a prognostic biomarker in Western Crohn's disease (CD) patients, and are associated with autophagy-associated ATG16L1 and NOD2 variants. We hypothesized that genetic determinants of Paneth cell phenotype in other ethnic CD cohorts are distinct but also involved in autophagy. METHODS. We performed a hypothesis-driven analysis of 56 single nucleotide polymorphisms (SNPs) associated with CD susceptibility or known to affect Paneth cell function in 110 Japanese CD patients who underwent ileal resection. We subsequently performed a genome-wide association analysis. Paneth cell phenotype was determined by defensin-5 immunofluorescence. Selected genotype-Paneth cell defect correlations were compared to a Western CD cohort (n = 164). RESULTS. The average percentage of abnormal Paneth cells in Japanese CD was similar to Western CD (P = 0.87), and abnormal Paneth cell phenotype was also associated with early recurrence (P = 0.013). In contrast to Western CD, ATG16L1 T300A was not associated with Paneth cell defect in Japanese CD (P = 0.20). Among the 56 selected SNPs, only LRRK2 M2397T showed significant association with Paneth cell defect (P = 3.62 × 10-4), whereas in the Western CD cohort it was not (P = 0.76). Pathway analysis of LRRK2 and other candidate genes with P less than 5 × 10-4 showed connections with known CD susceptibility genes and links to autophagy and TNF-α networks. CONCLUSIONS. We found dichotomous effects of ATG16L1 and LRRK2 on Paneth cell defect between Japanese and Western CD. Genes affecting Paneth cell phenotype in Japanese CD were also associated with autophagy. Paneth cell phenotype also predicted prognosis in Japanese CD. FUNDING. Helmsley Charitable Trust, Doris Duke Foundation (grant 2014103), Japan Society for the Promotion of Science (KAKENHI grants JP15H04805 and JP15K15284), Crohn's and Colitis Foundation grant 274415, NIH (grants 1R56DK095820, K01DK109081, and UL1 TR000448).

Target Intestinal Microbiota to Alleviate Disease Progression in Amyotrophic Lateral Sclerosis

PurposeEmerging evidence has demonstrated that gut microbiome plays essential roles in the pathogenesis of human diseases in distal organs. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Treatment with the only drug approved by the US Food and Drug Administration for use in ALS, riluzole, extends a patient׳s life span by only a few months. Thus, there is an urgent need to develop novel interventions that for alleviate disease progression and improve quality of life in patients with ALS. Here we present evidence that intestinal dysfunction and dysbiosis may actively contribute to ALS pathophysiology. MethodsWe used G93A transgenic mice as a model of human ALS. The G93A mice show abnormal intestinal microbiome and damaged tight junctions before ALS disease onset. The mice were given 2% butyrate, a natural bacterial product, in the drinking water. ResultsIn mice fed with butyrate, intestinal microbial homeostasis was restored, gut integrity was improved, and life span was prolonged compared with those in control mice. At the cellular level, abnormal Paneth cells—specialized intestinal epithelial cells that regulate the host–bacterial interactions—were significantly decreased in the ALS mice treated with butyrate. In both ALS mice and intestinal epithelial cells cultured from humans, butyrate treatment was associated with decreased aggregation of the G93A superoxide dismutase 1 mutated protein. ImplicationsThe findings from this study highlight the complex role of the gut microbiome and intestinal epithelium in the progression of ALS and present butyrate as a potential therapeutic reagent for restoring ALS-related dysbiosis.

Foxa2 and Hif1ab regulate maturation of intestinal goblet cells by modulating agr2 expression in zebrafish embryos.

Mammalian anterior gradient 2 (AGR2), an endoplasmic reticulum (ER) protein disulfide-isomerase (PDI), is involved in cancer cell growth and metastasis, asthma and inflammatory bowel disease (IBD). Mice lacking Agr2 exhibit decreased Muc2 protein in intestinal goblet cells, abnormal Paneth cell development, ileitis and colitis. Despite its importance in cancer biology and inflammatory diseases, the mechanisms regulating agr2 expression in the gastrointestinal tract remain unclear. In the present study, we investigated the mechanisms that control agr2 expression in the pharynx and intestine of zebrafish by transient/stable transgenesis, coupled with motif mutation, morpholino knockdown, mRNA rescue and ChIP. A 350bp DNA sequence with a hypoxia-inducible response element (HRE) and forkhead-response element (FHRE) within a region -4.5 to -4.2kbp upstream of agr2 directed EGFP expression specifically in the pharynx and intestine. No EGFP expression was detected in the intestinal goblet cells of Tg(HREM:EGFP) or Tg(FHREM:EGFP) embryos with mutated HRE or FHRE, whereas EGFP was expressed in the pharynx of Tg(HREM:EGFP), but not Tg(FHREM:EGFP), embryos. Morpholino knockdown of foxa1 (forkhead box A1) reduced agr2 levels in the pharynx, whereas knockdown of foxa2 or hif1ab decreased intestinal agr2 expression and affected the differentiation and maturation of intestinal goblet cells. These results demonstrate that Foxa1 regulates agr2 expression in the pharynx, whereas both Foxa2 and Hif1ab control agr2 expression in intestinal goblet cells to regulate maturation of these cells.

Effects of combined exposure to aluminum chloride and γ-radiation on histological and ultrastructure of intestinal Paneth cells

PurposeThe use of aluminum chloride (AlCl3) as in purification of water and the exposure to ionizing radiation (IR) in therapeutic treatments are believed to be relatively safe. However, their combined effects remain unclear. The aim of the present study is to investigate the effects of concomitant exposure to aluminum chloride and gamma radiation on the histological and ultra-structural pattern of the crypt Paneth cells (PC) of the small intestine in albino rats. Materials and methodsForty male albino rats were divided randomly into four groups (n = 10). Group 1: Control group, Group 2: Aluminum treated group (Rats received a daily a dose of 0.5 mg of AlCl3 per kg of body weight/day), Group 3: γ-Radiation group (rats are exposed to a total dose of 8 Gy in 4 fractions of 2 Gy/week) and Group 4 Rats exposed to AlCl3 and exposed to γ-Radiation. Animals were sacrificed after 4 weeks, one day after the last radiation dose. ResultsHistological and ultra-structural studies in small intestine showed that exposure to γ-rays induced alterations in PC including apoptotic nuclei, presence of injury in secretory granules and completely damaged organelles at the sites of bacterial translocation in the crypt of lumens. Aluminum exposure during irradiation potentiate the damage notified by abnormal PC morphology, dilation the crypt's lumen and erosions of its villi with increased apoptosis in the crypt cells and appearance of homogenously electron-lucent granules. ConclusionsIt is concluded that concomitant exposure to aluminum and IR increased detrimental structural changes in PC of rat intestine due to their combined effect. It is thus recommended to limit the intake of aluminum when human are at risk of over exposure to ionizing radiation.

VDR/vitamin D receptor regulates autophagic activity through ATG16L1

ABSTRACTThe Paneth cell is a unique intestinal epithelial cell that can sense the gut microbiome and secrete anti-microbial peptides, thereby playing critical roles in the maintenance of homeostasis at the intestinal-microbial interface. These roles in regulating innate immunity and intestinal microbial ecology are dependent on a functional autophagy pathway through ATG16L1. ATG16L1 is a regulator for autophagy and a risk gene for inflammatory bowel disease (IBD). We demonstrated that a low VDR/vitamin D receptor level in the intestine is associated with abnormal Paneth cells, impaired autophagy function, and imbalanced bacterial profile (dysbiosis), accompanied by a reduction of ATG16L1. We determined that VDR transcriptionally regulates ATG16L1 as a VDR target gene. Administration of the bacterial product butyrate increases intestinal VDR expression and suppresses inflammation in a colitis model. Thus, our study indicates that VDR may be a determinant of IBD risk through its actions on ATG16L1. These insights can be leveraged to define therapeutic targets for restoring Paneth cells and autophagy through VDR in chronic inflammation. It may also have applicability for infectious diseases and autoimmune diseases associated with skin or lung, where the host is in contact with bacteria.

O-011 Paneth Cell Phenotypes Define a Subtype of Pediatric Crohnʼs Disease Through Alterations in Host-Microbial Interactions

To identify and develop novel targeted therapies for complex diseases such as Crohn's disease (CD), functional subtypes rooted in pathogenesis must be defined. One candidate method to subtype CD is to define the small intestinal Paneth cell phenotypes based on the intracellular distribution of antimicrobial proteins. We have previously shown that in CD patients, Paneth cell phenotype correlates with the patients' genotype, distinct gene expression signature, presence of granuloma (pathologic hallmark), and time to recurrence after surgery. However, the mechanism by which abnormal Paneth cells contribute to pathogenesis and sub-classify disease in the context of host-microbial interaction is unclear. We analyzed Paneth cell phenotype and its correlation with mucosal microbiome and transcriptome in a cohort of pediatric CD and non-inflammatory bowel disease (IBD) patients.We first retrospectively analyzed Paneth cell phenotypes using archived resection specimens from adult (n = 531) and pediatric (n = 73) CD patients. We next analyzed a prospectively recruited pediatric cohort, including CD (n = 44) and non-IBD (n = 62) patients aged 4 to 18. These patients were recruited at the time of routine endoscopy. Ileal mucosal biopsy samples were collected and analyzed for Paneth cell phenotype (lysozyme/defensin 5 immunofluorescence), mucosal microbiome (16S rRNA sequencing), and transcriptome (RNA-sequencing). Paneth cell phenotype was determined by the percentage of normal Paneth cells in each sample. Type I Paneth cell phenotype was defined as <80% normal Paneth cells, whereas Type II Paneth cell phenotype was defined as ≥80% normal Paneth cells.The prevalence of type I Paneth cell phenotype in pediatric CD patients was higher than in adult CD cohorts (47% versus 18%; P < 0.0001). In pediatric CD patients, the type I Paneth cell phenotype was associated with significant changes in the ileal mucosal microbiome, characterized by reduced abundance of barrier-associated microbes (Faecalibacterium, Blautia, Ruminococcaceae, Porphyromonas, Lachnospira, Peptostreptococcus, Anaerostipes, and Odoribacteraceae) and enrichment of potentially pro-inflammatory microbes (Corynebacterium and Erysipelotrichaceae). In addition, pediatric CD patients with type I Paneth cell phenotype also displayed an altered epithelial gene expression profile, with significant reduction in oxidative phosphorylation gene cluster. Furthermore, the down regulation of oxidative phosphorylation gene cluster in CD was in turn associated with reduced abundance of Faecalibacterium, suggesting a complex network between Paneth cell function, epithelial energy/metabolism, and microbiome homeostasis. The connections between Paneth cell phenotypes, microbiome, and transcriptome profiles were not observed in non-IBD patients.These data support a functional role for Paneth cells in subtyping CD that is based on a specific pattern of metabolic dysregulation and mucosal dysbiosis.